Clinic‐ready inhibitor of MMP‐9/‐12 restores sensory and functional decline in rodent models of spinal cord injury

This study demonstrated that short-term inhibition of matrix metalloprotease (MMP)-9 and MMP-12 in both mouse and rat models of spinal cord injury (SCI) using the clinic-ready, orally bioavailable and specific inhibitor, AZD1236, attenuated injury-induced oedema, proinflammatory pain markers, pain sensation and blood-spinal cord barrier (BSCB) breakdown. Inhibition of MMP-9 and MMP-12 also protected against SCI-induced sensory and locomotor deficits. By demonstrating these unprecedented improvements with a clinic-ready MMP inhibitor, using a dosing regimen, which is anticipated to be safe and well tolerated in SCI patients, we are now well-placed to move swiftly into a Phase 2a study in patients.

AZD1236 treatment promoted significant axon regeneration ( Figure 4A,B) compared to other MMP inhibitors ( Figure S5) and increased neurofilament 200 (NF200) + spared fibres in the spinal cord above (T9) and below (T7) the lesion ( Figure 4C,D). CSF and serum biomarkers of SCI were also significantly attenuated by AZD1236 ( Figure S6A-E). Axon regeneration and enhanced sparing of axons after SCI correlated with improved electrophysiological function ( Figure 4E-G) and significant improvements in locomotor ( Figure 4H) and sensory function ( Figure 4I), with intrathecal delivery of AZD1236 F I G U R E 1 Matrix metalloprotease (MMP)-9 and MMP-12 levels and their enzymatic activity increase acutely after dorsal column (DC) injury in mice, and AZD1236 significantly suppresses MMP-9 and MMP-12 activity. (A) Levels of MMP-9 mRNA peak 1 day after injury. (B) Levels of MMP-12 mRNA peak at 5 days after injury. (C) MMP-9 protein levels also peak 1 day after injury. (D) MMP-12 protein levels also peak at 5 days after injury. (E) MMP-9 activity is high at 1 day and peaks by 3 days after injury. (F) MMP-12 activity peaks at 5 days after injury. (G) MMP-9 activity is suppressed by oral delivery of AZD1236 for 3 days after injury in both serum and cerebrospinal fluid (CSF). (H) MMP-12 activity is also suppressed by oral delivery of AZD1236 in both serum and CSF. (I) MMP-9 activity is suppressed by intrathecal delivery of AZD1236 for 3 days after injury in both serum and CSF. (J) MMP-12 activity is also suppressed by intrathecal delivery of AZD1236 in both serum and CSF. (K) Oral delivery of AZD1236 for 3 days after injury suppresses MMP-9 activity by 90% in both serum and CSF, whilst MMP-12 is suppressed by 74% and 69% in CSF, respectively. Intrathecal delivery of AZD1236 for 3 days after injury suppresses MMP-9 activity by 71% in both serum and CSF, whilst MMP-12 activity is suppressed by 88 and 90% in serum and CSF, respectively. n = 6 mice/group. (L) Optimal doses of AZD1236 also significantly suppress MMP-9 and MMP-12 activity in spinal cord homogenates at 3 days after injury. RFU = relative fluorescence units. (M) In situ zymography in saggital sections of the lesion site at 3 days after injury shows that the high levels of gelatinase activity (green; arrowheads) after DC injury is suppressed after oral and intrathecal delivery of optimal doses of AZD1236 in spinal cord sections. Sections are counterstained with GFAP (red) to mark astrocytes in red. # = lesion site. Data are expressed as means ± standard error of the mean (SEM). n = 6 mice/group, two independent experiments, total n = 12 mice/group. p = .0001, one-way analysis of variance (ANOVA) with Dunnett's post hoc test. Scale bars in (M) = 200 μm. NOTE: AZD1236 treatment was provided immediately after injury having similar benefits ( Figure 4J-N). Moreover, oral and intrathecal AZD1236 treatment in the severe CC injury model at the same lowest effective dose as the DC model, also significantly improved electrophysiological function across the lesion site ( Figure S7A-C) and locomotor performance ( Figure S7D,E).
Even with a 24-h delay to treatment after SCI, optimal doses of AZD1236 were equally as effective as immediate delivery, in suppressing SCI-induced water content ( Figure S8A), proinflammatory cytokines ( Figure S8B), MMP-9 and MMP-12 activity ( Figure S8C) and improved electrophysiological ( Figure S8D-F), F I G U R E 2 Inhibition of matrix metalloprotease (MMP)-9 and MMP-12 using AZD1236 ablates spinal cord injury (SCI)-induced oedema at 3 days after dorsal column (DC) injury in mice and suppresses proinflammatory pain markers and behavioural correlates of pain. (A) 200 mg/kg of AZD1236 effectively ablated SCI-induced oedema by oral delivery. (B) 5 mg/kg of AZD1236 effectively ablated SCI-induced oedema by intrathecal delivery. Data are expressed as means ± SEM. n = 6 mice/group, three independent experiments, total n = 18 mice/group. (C) Inhibition of both MMP-9 and MMP-12 is required to ablate SCI-induced oedema. Note: MMP408 and Inhibitor I were used at doses above when used singly, however, when combined doses are halved (i.e., 100 mg/kg = 50 mg/kg MMP408/50 mg/kg Inhibitor I, 200 mg/kg = 100 mg/kg MMP408/100 mg/kg Inhibitor I, 300 mg/kg = 150 mg/kg MMP408/150 mg/kg Inhibitor I). Data are expressed as means ± SEM. n = 6 mice/group, two independent experiments, total n = 12 mice/group. **p = .01; ***p = .0001, one-way ANOVA with Dunnett's post hoc test. NOTE: AZD1236 treatment was provided immediately after injury. (D) Inhibition of MMP-9 and MMP-12 by oral AZD1236 attenuates mRNA levels of proinflammatory pain markers interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the DC injury model. locomotor ( Figure S8G) and sensory function ( Figure S8H) as well as axon regeneration ( Figure S9A,B). At present, we are unsure why delayed treatment is as effective as immediate treatment but believe that there is a time window of therapeutic value of AZD1236 to counteract the negative effects of acute, dysregulated MMP-9 and MMP-12 activity.
Since the rat DC injury model better recapitulates human SCI pathophysiology, 1,5 the same profile of MMP-9 ( Figure S10A) and MMP-12 ( Figure S10B) mRNA expres-sion was also observed. AZD1236 is inactive against rat MMP-9/-12, and hence AZD3342, which has similar selectivity to AZD1236, but is active in the rat, demonstrated that SCI-induced oedema ( Figure S10C), MMP-9 and MMP-12 activity ( Figure S10D), and proinflammatory pain cytokines ( Figure S10E) could also be suppressed in the rat DC injury model accompanied by significantly improved electrophysiological ( Figure S10F), locomotor ( Figure S10G) and sensory function ( Figure S10H), similar to that observed with in the mouse SCI model. Finally, Quantification of the number of NF200 pixels above and below the lesion site to demonstrate sparing of axons in DC+AZD1236-treated animals compared to DC+vehicle-treated controls. Data are expressed as means ± SEM. n = 6 mice/group, two independent experiments, total n = 12 mice/group. ***p = .0001, one-way ANOVA with Dunnett's post hoc test. (E) Representative Spike 2 processed compound action potential (CAP) traces after oral delivery of AZD1236 showing ablation of CAP waves at 6 weeks after DC injury but restoration of a significant CAP wave by oral AZD1236. (F) Oral AZD1236 significantly improved CAP amplitudes at 6 weeks after DC injury. (G) Oral AZD1236 significantly improved CAP areas at 6 weeks after DC injury. (H) Oral AZD1236 significantly improved ladder crossing performance (locomotor function) at 6 weeks after DC injury. (I) Oral AZD1236 significantly improved tape sensing and removal performance (sensory function) over 6 weeks after DC injury. (J) Representative Spike 2 processed CAP traces after intrathecal delivery of AZD1236 showing ablation of CAP waves over 6 weeks after DC injury but restoration of a significant CAP wave by intrathecal AZD1236. (K) Intrathecal AZD1236 significantly improved CAP amplitudes at 6 weeks after DC injury. (L) Intrathecal AZD1236 significantly improved CAP areas at 6 weeks after DC injury. (M) Intrathecal AZD1236 significantly improved ladder crossing performance (locomotor function) over 6 weeks after DC injury. (N) Intrathecal AZD1236 significantly improved tape sensing and removal performance (sensory function) over 6 weeks after DC injury. Data are expressed as means ± SEM. n = 6 mice/group, three independent experiments, total n = 18 mice/group. ***p = .0001, one-way ANOVA with Dunnett's post hoc test. § p = .0001, independent sample t-test. # p = .0015, linear mixed models; ## p = .0011, generalized linear mixed models. NOTE: AZD1236 treatment was provided immediately after injury since we advocate short-term inhibition (i.e., the first 3 days after SCI) of MMP-9 and MMP-12, we showed that MMP-9 took 4 days, whilst MMP-12 took 5 days to return to normal SCI-induced levels in the injury site, once AZD1236 is withdrawn ( Figure S11A,B).
In conclusion, we showed that AZD1236, administered within 24 h after SCI and for only 3 days, promotes unequivocal positive benefits to the key pathophysiological consequences of SCI. 6-10 AZD1236 suppresses SCI-induced oedema, BSCB breakdown, neuropathic pain, scarring and infiltration of macrophages into the lesion site while at the same time promoting axon regeneration, leading to improvements in electrophysiological, sensory and locomotor function. This is potentially the first treatment for SCI that is capable of promoting such unprecedented benefits.

A C K N O W L E D G E M E N T S
The authors thank AstraZeneca (UK) for providing AZD1236/AZD3342 under a material transfer agreement through the AstraZeneca Open Innovation program. They apologize to colleagues whose works related to MMP inhibitors we did not cite in this manuscript.

C O N F L I C T O F I N T E R E S T
Rebecca J. Fairclough is an employee of AstraZeneca UK. Zubair Ahmedis an inventor on a patent related to this work. All other authors declare that they have no competing interests.